IP problem: antibody binds too few antigen - (Feb/19/2009 )
I'm trying to establish IP in our lab. My problem is that after IP my IgG band is much stronger then the antigen band. If IP would be perfect, then should't the antigen band be about as strong as the IgG band? (I use the same antibody for IP as for the subsequent Western blot).
I ran on my gel the original lysate (that has not been immunoprecipitated) and the supernatant of the IP reaction too, and in the IP supernatant there is as much antigen as in the unprecipitated lysate. And the solute eluted from the beads (that is the precipitated sample) contains as much (or even a little less) antigen as the unprecipitated sample in spite that the immunoprecipitated sample must be much more concentrated then the unprecipitated one.
I tried to reduce the ammount of the antibody, but in that case both, the IgG and the antigen band were lowered in the IP sample. Why does not bind my antibody more antigen? (The antibody I use is very good.)
Any answer would be appreciated.
About concentration by IP, I cannot answer.
but if you don't want to see the Ig band in your western, you should use a HRP-conjugated protein G instead of the HRP-conjugated anti Ig. protein G will mainly recognize the native primary antibody you use to detect the antigen, but not the denatured Ig that are loaded on the gel.
My problem is not the presence of the IgG band, but the small size of the antigen band compared to the IgG band.
Nevertheless, later the presence of the IgG band might be a factor to consider, and so thanks for the idea. So far I have thought of crosslinking as a method to eliminate the IgG band, but this other method might work as well.
So the question is: why doesn't my antibody bind more antigen?
from my experience, I never had a concentration of my antigen by IP.( It was not the goal. )
When I want to purify an antigen, I dilute more my sample, and I load the sample on a column where the antigen is covalently linked. the antigen is eluted by decreasing the pH.
I don't know if others have a concentration of their antigen after IP?
Well, I just thought that I should get about as strong antigen band in my IP as the IgG band (or a little lower as not all of the antibodies bind antigen of course). But my IgG band is much stronger than the antigen band. And the supernatant of the IP reaction (that is the solution remaining after IP) has as strong antigen band as the control (not immunoprecipitated) sample. I don't know if it is normal or not.
What is the species of your primary?
Is it the same as your secondary?
If so, it is likely anything you see is an artifact. Primary and secondary
antibodies really should be different species. This is especially true for Rabbit and Goat antibodies.
What size is your protein?
Why do you think the amount of your IPed protein would be equal intensity to your IgG band?
This is utter nonsense. Your IgG band will always be stronger. You antibody may not be very efficient.
Yes, all my primary antibodies are of rabbit origin. I thought that one molecule of antibody binds one molecule of antigen. Though not all the antibody binds antigen, I thought that their intensity must be around equal. But thinking again it is very reasonable that only a portion of the antibody binds antigen. I simply didn't know what to expect. So does protein A have a higher affinity to the antibody as the antibody has to the antigen? I had no information about it at all.
About artefacts: I hope, it is not. I have a band at around 50 kDa (it must be the IgG, as it is missing from the control sample and more importantly it is missing from the non precipitated sample). The other band is a bit higher and it seems to be O.K., as the antigen is protein kinase B (Akt), whose mol weight is aroun 56 kDa.
In the non-immunoprecipitated sample (and in the supernatant of the IP reaction) two strong bands are present (the higher one a bit stronger but the lower one a bit more compact) while in the IP samples only the lower, more compact band can be seen (and the IgG of course). It is important that first I tried to catch estrogen receptor alpha, but failed. In that case I saw the IgG band only. So I hope that my other band in the last IP recations is the Akt.
I used 400 mcl lysate for the IP reactions and at the end eluted the beads in a volume of 80 mcl (including the beads). Non precipitated samples were the original lysates diluted with 2x sample buffer. And after Western blot I see that the IP reaction contains as much (or a little lower) ammount of Akt as the non-precipitated samples. It is not what I expected.
Could I improve my antigen yeald if I first incubated the protein A-agarose beads with antibody and then added these beads to my lysate? My idea is that in this way I could raise the local concentration of the antibody on the surface of the beads.
leptopelis on Feb 19 2009, 09:58 PM said:
protein-antigen interaction has the higher affinity
leptopelis on Feb 20 2009, 09:04 AM said:
I never tried this way.
normally you have an excess of antibody compared to antigen, and an excess of beads compared to antibodies, to be sure to IP as much of antigen as possible (that's why you will always have a higher band for antibodies than for antigens.
if you incubate first the beads and antibody, one could say that you could have a sterical "encombrement" that doesn't allow the antigen to bind to the antibody; or not so well.
I don't know. Have a try